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A Reference Library of DNA Barcodes for Nordic Lichens

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A Reference Library of DNA Barcodes for Nordic Lichens Biodiversity Data Journal 7: e36252 doi: 10.3897/BDJ.7.e36252 Data Paper OLICH: A reference library of DNA barcodes for Nordic lichens Gunnhild Marthinsen‡‡, Siri Rui , Einar Timdal‡ ‡ Natural History Museum, University of Oslo, Oslo, Norway Corresponding author: Gunnhild Marthinsen ([email protected]) Academic editor: Andreas Beck Received: 16 May 2019 | Accepted: 31 Jul 2019 | Published: 19 Aug 2019 Citation: Marthinsen G, Rui S, Timdal E (2019) OLICH: A reference library of DNA barcodes for Nordic lichens. Biodiversity Data Journal 7: e36252. https://doi.org/10.3897/BDJ.7.e36252 Abstract Background DNA barcodes are increasingly being used for species identification amongst the lichenised fungi. This paper presents a dataset aiming to provide an authoritative DNA barcode sequence library for a wide array of Nordic lichens. New information We present 1324 DNA barcode sequences (nrITS) for 507 species in 175 genera and 25 orders. Thirty-eight species are new to GenBank and, for 25 additional species, ITS sequences are here presented for the first time. The dataset covers 20–21% of the Nordic lichenised species. Barcode gap analyses are given and discussed for the three genera Cladonia, Ramalina and Umbilicaria. The new combination Bryobilimbia fissuriseda (Poelt) Timdal, Marthinsen & Rui is proposed for Mycobilimbia fissuriseda and Nordic material of the species, currently referred to as Pseudocyphellaria crocata and Psoroma tenue ssp. boreale, are shown to belong in Pseudocyphellaria citrina and Psoroma cinnamomeum, respectively. © Marthinsen G et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Marthinsen G et al Keywords DNA barcoding, ITS, Lichenised fungi, Norway, Taxonomy Introduction Lichens are fungi living in mutualistic symbiosis with green algae and/or cyanobacteria. They are highly polyphyletic and 20–30 independent lichenisation events may have occurred during the evolution of higher Fungi (Schoch et al. 2009, Lücking et al. 2017a). The lichenised fungi currently consists of more than 19,000 species, i.e. about 17% of all known fungal species and 27% of all known ascomycete species (Lücking et al. 2017a). They occur globally in all major terrestrial ecosystems, especially on trees, soil and rock. Although a lichen thallus consists of a symbiotic association between a fungal exhabitant, the mycobiont and one or more photosynthetic algal and/or cyanobacterial inhabitants, the photobionts, the taxonomy and nomenclature follow that of the mycobiont. A lichen thallus may house additional fungi, i.e. parasites, parasymbionts and endophytes (Hawksworth 1982, Richardson 1999, Spribille et al. 2016), making a lichen thallus a complex symbiotic assemblage. Identifying lichenised fungi has traditionally been based on morphological, anatomical and chemical characters, but DNA-based identification is now increasingly being used. In 2012, the internal transcribed spacer of nuclear ribosomal DNA (ITS) was recommended as the primary fungal DNA barcode marker by the International Fungal Barcoding Consortium (Xu 2016), following studies on its suitability by for example Schoch et al. (2012). For lichenised fungi, studies reporting successful use of the ITS marker for species delimitations include Kelly et al. (2011), Zheng et al. (2011), Parnmen et al. (2013), Leavitt et al. (2014), Magain and Serusiaux (2015) and Divakar et al. (2016). Pino-Bodas et al. (2013), however, found ITS often failing for species delimitations in Cladonia and investigated alternative markers. In this paper, we present a DNA barcode dataset for Nordic lichens, including 1324 records, comprising 507 species in 175 genera and 25 different orders. General description Purpose: This dataset aims to provide an authoritative DNA barcode sequences library for a wide array of Nordic lichens. The library should facilitate DNA-based identification of species for both traditional molecular studies and DNA-metabarcoding studies and should also be a resource for biosystematic research on Nordic lichens. Additional information: Eighty sequences have been published previously, but are included here as they were produced under the OLICH project, i.e. GenBank IDs KT800002–07 (Westberg et al. 2015), KU687445–60 (McCune et al. 2016), KU873926–32 OLICH: A reference library of DNA barcodes for Nordic lichens 3 (Timdal et al. 2016), KY682961 (Pykälä et al. 2018), 38 sequences within the series MG838156–201 (Kistenich et al. 2018), MG968256–61 (Haugan and Timdal 2018) and MK567159–64 (Haugan and Timdal 2019). In addition, 151 sequences (KY266831–981) have been submitted to GenBank previously for an unpublished project. Results: Thirty-eight species are new to GenBank: Arthonia phaeobaea, Arthopyrenia salicis, Bactrospora corticola, Baeomyces carneus, Buellia arnoldii, B. dives, Candelariella arctica, Cladonia cyanipes, Coccotrema citrinescens, Eopyrenula leucoplaca, Epilichen glauconigellus, E. scabrosus, Gyalecta foveolaris, Lecanora atrosulphurea, L. leptacina, Ochrolechia alaskana, O. alboflavescens, O. gowardii, O. mahluensis, Orphniospora moriopsis, Phaeophyscia kairamoi, Pilophorus dovrensis, Pseudocyphellaria norvegica, Pyrenula occidentalis, Ramalina cuspidata, Rhizocarpon badioatrum, R. bolanderi, R. chioneum, R. jemtlandicum, R. leptolepis, R. rittokense, R. santessonii, R. subgeminatum, Rinodina balanina, R. disjuncta, R. roboris, Stereocaulon capitellatum and S. symphycheilum. Twenty-five additional species are previously represented in GenBank, but not with ITS sequences: Arthonia didyma, Bryobilimbia fissuriseda, Buellia epigaea, Callome multipartita, Dendrographa latebrarum, Fuscidea mollis, Gyalecta flotowii, G. friesii, G. geoica, Gyrographa gyrocarpa, Halecania alpivaga, Koerberiella wimmeriana, Opegrapha vermicellifera, Pertusaria aspergilla, P. carneopallida, Phlyctis agelaea, Placynthiella oligotropha, Psoronactis dilleniana, Pyrenula laevigata, Ramboldia elabens, Sagiolechia protuberans, Stereocaulon grande, Thelotrema macrosporum, Tremolecia atrata and Varicellaria lactea. Project description Title: The name OLICH refers to “contributions from the Oslo lichen herbarium”. Personnel: Gunnhild Marthinsen (project coordinator), Siri Rui (project engineer), Einar Timdal (taxonomic supervisor), all affiliated to the Natural History Museum, University of Oslo. Study area description: The Nordic countries, including Svalbard: Norway, mainland (97.0% of the specimens), Sweden (2.0%), Finland (0.5%), Norway, Svalbard (0.5%), Fig. 1. Funding: This project is funded by the Norwegian Research Council (RCN 226134) through the Norwegian Barcode of Life project (NorBOL). In addition, the Norwegian Species Information Centre (Artsdatabanken) has funded four projects on lichens under the Norwegian Taxonomy Initiative from which material has been collected and/or sampled (project numbers 70184216, 70184227, 70184233, and 70184237). 4 Marthinsen G et al Figure 1. Distribution of the OLICH records Sampling methods Sampling description: The material was partly freshly collected specimens from the field and partly previously curated specimens in university herbaria. The distribution of specimen age at seqencing is given in Fig. 2. The specimens were initially determined by experts based on morphology and secondary chemistry and the identifications were re- examined at the quality control after sequence acquisition. All collected material were, after sampling, deposited in university herbaria and the entire material is housed in BG (2.1%), H (0.1%), O (94.9%), SAV (0.2%), TRH (1.7%) and TROM (1.1%) (abbreviated according to Index Herbariorum; http://sweetgum.nybg.org/science/ih). Figure 2. Distribution of specimen age (years after collecting) at sequencing in the OLICH dataset. OLICH: A reference library of DNA barcodes for Nordic lichens 5 Thin-layer chromatography (TLC) was performed for identification of secondary compounds on selected specimens in accordance with the methods of Culberson (1972), as modified by Menlove (1974) and Culberson and Johnson (1982). Solvent system B' was used for routine examination, A and C for additional confirmation. Tissue of thallus or preferably apothecia (about 3 mm3 ) was assembled in 96 well plates obtained from the Canadian Centre for DNA Barcoding (CCDB; http://ccdb.ca). One well was left empty to serve as negative control. The freshly collected material was either sampled in the field or at a field station. In the field, the specimen was first photographed, then tissue was removed with a flame-decontaminated forceps and placed in the well and finally the specimen was collected. Specimens requiring more controlled handling were photographed and sampled either with forceps or scalpel under a binocular lens at a field station. Wells containing fresh material were dried for several days with lids open in a drying chamber containing silica gel. Herbarium material was sampled at the housing institutions under binocular lenses. Field data was stored on tablets and GPSs. Tissue sampling data and photography data were transferred directly to standard BOLD spreadsheets, whereas the collection data passed through herbarium databases for obtaining institution storing, voucher ID (= sample ID), uniform nomenclature and taxonomical hierarchy and contact information. Material sampled in herbaria used a similar pipeline, although simpler, as
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